Transcription factors AP-2α and AP-2β regulate distinct segments of the distal nephron in the mammalian kidney

Transcription factors AP-2α and AP-2β have been suggested to regulate the differentiation of nephron precursor populations towards distal nephron segments. Here, we show that in the adult mammalian kidney AP-2α is found in medullary collecting ducts, whereas AP-2β is found in distal nephron segments except for medullary collecting ducts. Inactivation of AP-2α in nephron progenitor cells does not affect mammalian nephrogenesis, whereas its inactivation in collecting ducts leads to defects in medullary collecting ducts in the adult. Heterozygosity for AP-2β in nephron progenitor cells leads to progressive distal convoluted tubule abnormalities and β-catenin/mTOR hyperactivation that is associated with renal fibrosis and cysts. Complete loss of AP-2β in nephron progenitor cells caused an absence of distal convoluted tubules, renal cysts, and fibrosis with β-catenin/mTOR hyperactivation, and early postnatal death. Thus, AP-2α and AP-2β have non-redundant distinct spatiotemporal functions in separate segments of the distal nephron in the mammalian kidney.

Cortical CDs (Aqp2 + ; blue arrows) and CTs (Calb1 + Aqp2 + ; red arrows) appear normal in 9-months-old Aqp2Cre + Tfap2a fl/fl mice, as seen in WT littermates. SMA labeling (yellow arrows) identifies pericytes of vessels in the kidney but no tubulointerstitial fibrosis is observed (mouse IgG deposits in vessel walls cause signal for the secondary anti-mouse antibody used to detect Calb1). Scale bars, 200μm.  Left: Reduction in body weight (BW) after 24-hour water deprivation is not significantly different between 3 months-old Aqp2Cre + Tfap2a fl/fl mice compared to WT littermates. Left middle: No significant difference is observed in 24-hour urine production in the presence or absence of water (water deprivation for 24 hours). Right middle: No difference in urine osmolality is observed between 3-months-old or 9-months-old Aqp2Cre + Tfap2a fl/fl mice and WT littermates. Right: No difference in BW in 5-8-months-old Aqp2Cre + Tfap2a fl/fl mice compared to WT littermates irrespective of gender. P-values are shown (two-tailed t-test). Graphs represent data as mean ± SEM. Number of mice per group is indicated in brackets. Source data are provided as a Source Data File. B.
No significant differences in BUN levels or 24-hour urine production or water intake in 5-8-months-old Aqp2Cre + Tfap2b fl/fl mice. P-values are shown (two-tailed Mann Whitney test). Graphs represent data as mean ± SEM. Number of mice per group is indicated in brackets. Source data are provided as a Source Data File. C.
Normal histology of 10-12-months-old Aqp2Cre + Tfap2b fl/fl mice compared to WT littermate mice. H&E sections (left: whole kidney; right: high magnification images of the same kidney) Scale bars, 1mm left and 50μm right. D.

Figure S10
A. Left: Kidneys of Six2Cre + Tfap2b fl/fl mice and Six2Cre + Tfap2b fl/WT mice show an extensive leukocytic (CD45 + ) interstitial inflammatory infiltrate (yellow arrows). These mice show cortical cysts with detached epithelial cells in the lumen (CD45 -) (white arrows). Middle: Immunolabeling for the proliferation marker Ki67 is observed in some epithelial cells of dilated tubules/cysts (yellow arrows) in kidneys of both Six2Cre + Tfap2b fl/fl mice and Six2Cre + Tfap2b fl/WT mice. Right: Labeling of these kidneys with the lectin WGA (demarcates distal nephron tubules and glomeruli) and phalloidin (identifies PTs), as well as for CD133 (marks Bowman's capsule of glomeruli and PTs). Cortical cysts in Six2Cre + Tfap2b fl/fl mice and Six2Cre + Tfap2b fl/WT mice are not PTs (CD133 -, no phalloidin + brush membrane) (white arrows), albeit some PTs show extensive dilatation (yellow arrow). Protein casts are observed in dilated tubules (green arrows). Scale bars, 100μm. B.
Immunolabeling shows that DCTs in PvalbCre + Tfap2b fl/fl mice still express NCC (orange arrows). SMA labeling is detected in pericytes of vessels (white arrowheads) but no interstitial fibrosis is observed. 16-months-old mice. Scale bars, 100μm. C.
Reduced NCC protein levels in whole kidney lysates of 12-months-old PvalbCre + Tfap2b fl/fl mice compared to WT littermates (n=3 mice/group). Densitometric values for Western blot bands normalized to b-actin are shown. Size markers are indicated. P-value was determined by a two-tailed t-test. Graph represents data as mean ± SEM. Source data are provided as a Source Data File.  −log10 (FDR)   Reduced kidney size with distal nephron dilatation is seen in a P6 Six2Cre + Tfap2a fl/fl Tfap2b fl/fl mouse, whereas its Six2Cre + Tfap2a fl/fl Tfap2b fl/WT littermate does not show these defects. H&E staining. Scale bar, 500 μm. B.
Severe postnatal growth retardation in mice lacking AP-2b in the nephron proximal to the CDs, shown here in a direct comparison of a P6 Six2Cre + Tfap2a fl/fl Tfap2b fl/WT mouse with a Six2Cre + Tfap2a fl/fl Tfap2b fl/fl littermate. Images C.-E. are derived from these two mice. C.
Venn diagram of RNA-Seq data of whole kidney lysates from 2-months-old Six2Cre + Tfap2a fl/fl Tfap2b fl/WT mice compared to Cre-negative control littermates (n=3 mice/group) show reduced expression of genes that are normally highly expressed in DCTs, such as Pvalb and SFRP1 (arrows). DEGs: differentially expressed genes.  Whole kidney lysates of 13-15-months-old Six2Cre + Tfap2b fl/WT mice (n=5) and agematched WT controls (n=4) were used for Western blotting experiments, analyzing proteins as shown in Figure 4d. b-actin as a loading control. Densitometric values for Western blot bands normalized to b-actin are shown. Size markers are indicated by arrowheads. BUN values are shown for each mouse. pTFEB indicated by *, TFEB by *. B.
An increase in active and total b-catenin levels is observed in Six2Cre + Tfap2b fl/WT mice (n=5) compared to age-matched WT controls (n=4). P-values were determined by a two-tailed t-test. Graphs represent data as mean ± SEM. Source data are provided as a Source Data File. C. and D. A Six2Cre + Tfap2b fl/WT mouse with the highest BUN value (270.0) has also the highest increase in active b-catenin, associated with the greatest reduction in NCC and Slc3a1 (shown in A in red font). This inverse correlation between active b-catenin levels and NCC or Slc3a1 levels was consistently observed and shown with Pearson correlation analyses (Pearson r value, two-tailed p-value, and correlation matrices). Normalized to b-actin. N=5 mice/group.  Immunolabeling of vessels (CD31 + ) shows that the extensive glomerulosclerosis in aged Six2Cre + Tfap2b fl/WT mouse kidneys (8-months-old) leads to diminished glomerular tufts (yellow arrows), whereas age-matched WT or 1-months-old Six2Cre + Tfap2b fl/fl mouse kidneys show normal glomerular tufts (white arrows). Irregularly dilated vessels in tubulointerstitial areas of the renal cortex are observed in 1-months-old Six2Cre + Tfap2b fl/fl mouse kidneys (red arrow). Scale bars, 50 μm. AP-2a and AP-2b are expressed in the mouse epidermis and, thus, detection of immunolabeling in the mouse epidermis can provide validation of antibody specificity 2,3 . A. Immunolabeling with the anti-AP-2a antibody (rabbit anti-AP-2a; Abcam Cat# ab108311) detects nuclear AP-2a in keratinocytes of the mouse epidermis (white arrows) as well as in the dermal papilla (yellow arrows). Specificity of the immunolabeling is confirmed by the absence of AP-2a immunolabeling in keratinocytes from skin form mice in which AP-2a was inactivated in keratinocytes (Keratin14Cre + Tfap2a fl/fl mice). Immunolabeling in the dermal papilla persists in Keratin14Cre + Tfap2a fl/fl mice, which is not targeted by the Keratin14Cre, therefore, serving as a positive control for the immunolabeling within the same slide. B. Immunolabeling for AP-2b is detected in mouse keratinocytes (blue arrows) with the rabbit anti-AP-2b antibody (Cell Signaling Technology Cat# 2509). AP-2b immunolabeling is also detected in keratinocytes of Keratin14Cre + Tfap2a fl/fl mice, thereby ruling out potential crossreactivity of this antibody with AP-2a. C. Immunolabeling for AP-2b is also detected in mouse keratinocytes (blue arrows) with the anti-AP-2b antibody from Atlas (Atlas Antibodies Cat# HPA034683, RRID:AB_10670966), recapitulating the findings with the anti-AP-2b antibody from Cell Signaling Technology.
Scale bars, 50μm.    Table S2: Serum chemistries in experimental mouse groups of different ages. Chloride in mEq/l, potassium in mEq/l, sodium in mEq/l, magnesium in mg/dl, phosphorus in mg/dl, total protein in g/dl, albumin in g/dl, calcium in mg/dl, and globulin in g/dl. P-values are shown (two-tailed, unpaired t-test).